- Volume 150, Issue 11, 2004
Volume 150, Issue 11, 2004
- Review
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Nitrate reduction and the nitrogen cycle in archaea
More LessThe nitrogen cycle (N-cycle) in the biosphere, mainly driven by prokaryotes, involves different reductive or oxidative reactions used either for assimilatory purposes or in respiratory processes for energy conservation. As the N-cycle has important agricultural and environmental implications, bacterial nitrogen metabolism has become a major research topic in recent years. Archaea are able to perform different reductive pathways of the N-cycle, including both assimilatory processes, such as nitrate assimilation and N2 fixation, and dissimilatory reactions, such as nitrate respiration and denitrification. However, nitrogen metabolism is much less known in archaea than in bacteria. The availability of the complete genome sequences of several members of the eury- and crenarchaeota has enabled new approaches to the understanding of archaeal physiology and biochemistry, including metabolic reactions involving nitrogen compounds. Comparative studies reveal that significant differences exist in the structure and regulation of some enzymes involved in nitrogen metabolism in archaea, giving rise to important conclusions and new perspectives regarding the evolution, function and physiological relevance of the different N-cycle processes. This review discusses the advances that have been made in understanding nitrate reduction and other aspects of the inorganic nitrogen metabolism in archaea.
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- Microbiology Comment
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- Cell And Developmental Biology
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A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120
More LessThe impact of calcium signals in virtually all cells has led to the study of their role in prokaryotic organisms as stress response modulators. Cell differentiation in adverse conditions is a common Ca2+-requiring response. Nitrogen starvation induces the differentiation of N2-fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. This paper reports the use of a recombinant strain of this organism expressing the photoprotein aequorin to monitor the intracellular free-calcium concentration during the course of heterocyst differentiation. A specific calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and generated by intracellular calcium stores was identified. The intracellular calcium signal was manipulated by treatment with specific calcium drugs, and the effect of such manipulation on the process of heterocyst differentiation was subsequently assessed. Suppression, magnification or poor regulation of this signal prevented the process of heterocyst differentiation, thereby suggesting that a calcium signal with a defined set of kinetic parameters may be required for differentiation. A hetR mutant of Anabaena sp. PCC7120 that cannot differentiate into heterocysts retains, however, the capacity to generate the calcium transient in response to nitrogen deprivation, strongly suggesting that Ca2+ may be involved in a very early step of the differentiation process.
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- Biochemistry And Molecular Biology
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Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium
More LessThe structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N2 fixation, were identified and sequenced. 3′-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at −41·5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW.
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The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related
The Acinetobacter baumannii type strain, ATCC 19606, secretes acinetobactin, a catechol siderophore highly related to the iron chelator anguibactin produced by the fish pathogen Vibrio anguillarum (Listonella anguillarum). This paper reports the initial characterization of the genes and gene products involved in the acinetobactin-mediated iron-acquisition process. Insertional mutagenesis resulted in the isolation of several derivatives whose ability to grow in medium containing the iron chelator 2,2′-dipyridyl was affected. One of the insertions disrupted a gene encoding a predicted outer-membrane protein, named BauA, highly similar to FatA, the receptor for ferric anguibactin. Immunological relatedness of BauA with FatA was confirmed by Western blot analysis. Another transposon insertion was mapped to a gene encoding a protein highly similar to FatD, the permease component of the anguibactin transport system. Further DNA sequencing and nucleotide sequence analysis revealed that these A. baumannii 19606 genes are part of a polycistronic locus that contains the bauDCEBA ORFs. While the translation products of bauD, -C, -B and -A are highly related to the V. anguillarum FatDCBA iron-transport proteins, the product of bauE is related to the ATPase component of Gram-positive ATP-binding cassette (ABC) transport systems. This entire locus is flanked by genes encoding predicted proteins related to AngU and AngN, V. anguillarum proteins required for the biosynthesis of anguibactin. These protein similarities, as well as the structural similarity of anguibactin and acinetobactin, suggested that these two siderophores could be utilized by both bacterial strains, a possibility that was confirmed by siderophore utilization bioassays. Taken together, these results demonstrate that these pathogens, which cause serious infections in unrelated hosts, express very similar siderophore-mediated iron-acquisition systems.
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Autogenous modulation of the Bacillus subtilis sacB–levB–yveA levansucrase operon by the levB transcript
More LessSilencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis. Conversely, independent expression of levB greatly stimulates operon expression. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5′-AAAGCAGGCAA-3′) involved in the enhancing effect. In vitro, the levB transcript displays an affinity for the N-terminal fragment of SacY (K D 0·2 μM), the regulatory protein that prevents transcription termination of the levansucrase operon. This positive-feedback loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology.
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Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains
Members of the genera Streptococcus and Leuconostoc synthesize various α-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly α-(1→4) linkages together with α-(1→6) and α-(1→4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. This paper reports, for the first time, isolation and characterization of dextransucrase and mutansucrase genes and enzymes from various Lactobacillus species and the characterization of the glucan products synthesized, which mainly have α-(1→6)- and α-(1→3)-glucosidic linkages. The four GTF enzymes characterized from three different Lb. reuteri strains are highly similar at the amino acid level, and consequently their protein structures are very alike. Interestingly, these four Lb. reuteri GTFs have relatively large N-terminal variable regions, containing RDV repeats, and relatively short putative glucan-binding domains with conserved and less-conserved YG-repeating units. The three other GTF enzymes, isolated from Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri, contain smaller variable regions and larger putative glucan-binding domains compared to the Lb. reuteri GTF enzymes.
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A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II
Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.
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The C-terminal receiver domain of the Rhizobium leguminosarum bv. viciae FixL protein is required for free-living microaerobic induction of the fnrN promoter
More LessThe Rhizobium leguminosarum bv. viciae VF39 FixL protein belongs to a distinct group of hybrid regulatory sensor proteins that bear a covalently linked C-terminal receiver domain. FixL has an unorthodox histidine kinase domain, which is shared with many other hybrid regulators. The purified FixL protein had autophosphorylation activity. A truncated protein, lacking the receiver domain, had a much-reduced autophosphorylation activity. However, this truncated protein still efficiently phosphorylated the purified receiver domain in trans. This indicates that, in the full-length FixL protein, the conserved histidine residue in the kinase domain is phosphorylated only transiently and that most of the phosphoryl label accumulates in the C-terminal receiver domain. Gene-fusion studies showed that the fixL gene is required for free-living microaerobic induction of the fnrN promoter. The presence of a functional fixK gene is not required. An R. leguminosarum strain lacking fixL could not be complemented with a truncated copy of the gene lacking the receiver domain. This indicates that the C-terminal receiver domain is an intermediate in the signal transduction pathway that links oxygen limitation to induction of the fnrN promoter in R. leguminosarum bv. viciae VF39.
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Functional characterization of the BvgAS two-component system of Bordetella holmesii
More LessThe BvgAS two-component system is the master regulator of virulence gene expression in the mammalian pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. This paper reports the partial cloning and characterization of the bvgAS loci of the ‘new’ Bordetella species Bordetella holmesii, Bordetella trematum and Bordetella hinzii, which are increasingly recognized as opportunistic pathogens in humans. It is demonstrated that the cytoplasmic signalling domains of the BvgS histidine kinases of B. pertussis and B. holmesii are functionally interchangeable, while signal perception by the two sensor proteins seems to be different. Furthermore, it is shown that, despite the high similarity of the BvgA proteins of B. pertussis and B. holmesii, promoter recognition by the response regulator proteins differs substantially in these organisms.
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- Environmental Microbiology
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Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic–aerobic activated sludge processes
More LessIn an acetate-fed anaerobic–aerobic membrane bioreactor, a deteriorated enhanced biological phosphorus removal (EBPR) community was developed (as determined based on the chemical profiles of organic substrate, soluble phosphate, and intracellular carbohydrate and polyhydroxyalkanote (PHA) concentrations). Microscopic observations revealed the dominance of tetrad-forming organisms (TFOs), of which the majority stained positively for PHA under anaerobic conditions. Fluorescence in situ hybridization (FISH) confirmed that the Alphaproteobacteria (85·0±7·0 % of total cells) were the most dominant group. A 16S rRNA gene clone library specific for the Alphaproteobacteria indicated that most 16S rRNA gene clones (61 % of total clones) were closely affiliated with ‘Defluvicoccus vanus’, forming a cluster within subgroup 1 of the Alphaproteobacteria. Combined PHA staining and FISH with specific probes designed for the members of the ‘Defluvicoccus’ cluster suggested diversity within this TFO cluster, and that these TFOs were newly identified glycogen-accumulating organisms in EBPR systems. However, these ‘Defluvicoccus’-related TFOs were only seen in low abundance in 12 different EBPR and non-EBPR systems, suggesting that they were not the key populations responsible for the deterioration of full-scale EBPR processes.
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Novel organization of genes in a phthalate degradation operon of Mycobacterium vanbaalenii PYR-1
More LessMycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein (phtR) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit (phtAa), small subunit (phtAb), phthalate dihydrodiol dehydrogenase (phtB), phthalate dioxygenase ferredoxin subunit (phtAc) and phthalate dioxygenase ferredoxin reductase (phtAd). A 6·7 kb EcoRI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp.
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- Genes And Genomes
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The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation
The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.
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BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride
In fungi, phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. Due to its apparent simplicity, this response was chosen for pursuing photoreceptor isolation. Two genes were cloned, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, respectively. The BLR-1 protein has all the characteristics of a blue-light photoreceptor, whereas the structure of the deduced BLR-2 protein suggests that it interacts with BLR-1 through PAS domains to form a complex. Disruption of the corresponding genes demonstrated that they are essential for blue-light-induced conidiation. blr-1 and blr-2 were also shown to be essential for the light-induced expression of the photolyase-encoding gene (phr-1). Mechanical injury of mycelia was found to trigger conidiation of T. atroviride, a response not described previously. This response was not altered in the mutants. A novel effect of both red and blue light on mycelial growth was found involving another light receptor, which is compensated by the BLR proteins.
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Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria
More LessIn the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.
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The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1β group without any accessory genes
More LessThe nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1β backbone, which diverged early from the sequenced IncP-1β plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14·4 kb or 19·0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1β plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)–tetR–tnpA IS26 fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D β-lactamase (bla NPS-2); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)–tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1β plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1β plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1β plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.
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The complete genomes of Lactobacillus plantarum and Lactobacillus johnsonii reveal extensive differences in chromosome organization and gene content
The first comprehensive comparative analysis of lactobacilli was done by comparing the genomes of Lactobacillus plantarum (3·3 Mb) and Lactobacillus johnsonii (2·0 Mb). L. johnsonii is predominantly found in the gastrointestinal tract, while L. plantarum is also found on plants and plant-derived material, and is used in a variety of industrial fermentations. The L. plantarum and L. johnsonii chromosomes have only 28 regions with conservation of gene order, totalling about 0·75 Mb; these regions are not co-linear, indicating major chromosomal rearrangements. Metabolic reconstruction indicates many differences between L. johnsonii and L. plantarum: numerous enzymes involved in sugar metabolism and in biosynthesis of amino acids, nucleotides, fatty acids and cofactors are lacking in L. johnsonii. Major differences were seen in the number and types of putative extracellular proteins, which are of interest because of their possible role in host–microbe interactions. The differences between L. plantarum and L. johnsonii, both in genome organization and gene content, are exceptionally large for two bacteria of the same genus, emphasizing the difficulty in taxonomic classification of lactobacilli.
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Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system
Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1α plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla TLA-2 for a so far unknown Ambler class A extended spectrum β-lactamase. The integron-specific 3′-segment (qacEΔ1-sul1-orf5Δ) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2′-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second ‘genetic load’ region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.
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A virulence-associated gene microarray: a tool for investigation of the evolution and pathogenic potential of Staphylococcus aureus
More LessAn oligonucleotide probe microarray for investigation of the evolution of epidemic Staphylococcus aureus strains has been constructed. The array comprises 383 probes based on virulence-associated genes present in four key strains. Twelve strains including seven for which the complete chromosomal nucleotide sequence was available were tested on the array. Twenty-six per cent of the probes were able to differentiate between strains to give a minimum of two gene differences between pairs. A gene difference distance tree based on the array data had approximately the same topology as one prepared using concatenated MLST sequences. Differences in the topologies of these trees were found to indicate that large-scale recombination events had occurred during the evolution of the species. One such occurrence appears to have been a key event in the genesis of the EMRSA-15 clone (ST22) that currently represents the most prevalent methicillin-resistant S. aureus (MRSA) in the UK.
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Characterization of a dam mutant of Haemophilus influenzae Rd
More LessThe gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for CamR transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.
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Reconstruction of the evolutionary history of the LexA-binding sequence
More LessIn recent years, the recognition sequence of the SOS repressor LexA protein has been identified for several bacterial clades, such as the Gram-positive, green non-sulfur bacteria and Cyanobacteria phyla, or the ‘Alphaproteobacteria’, ‘Deltaproteobacteria’ and ‘Gammaproteobacteria’ classes. Nevertheless, the evolutionary relationship among these sequences and the proteins that recognize them has not been analysed. Fibrobacter succinogenes is an anaerobic Gram-negative bacterium that branched from a common bacterial ancestor immediately before the Proteobacteria phylum. Taking advantage of its intermediate position in the phylogenetic tree, and in an effort to reconstruct the evolutionary history of LexA-binding sequences, the F. succinogenes lexA gene has been isolated and its product purified to identify its DNA recognition motif through electrophoretic mobility assays and footprinting experiments. After comparing the available LexA DNA-binding sequences with the F. succinogenes one, reported here, directed mutagenesis of the F. succinogenes LexA-binding sequence and phylogenetic analyses of LexA proteins have revealed the existence of two independent evolutionary lanes for the LexA recognition motif that emerged from the Gram-positive box: one generating the Cyanobacteria and ‘Alphaproteobacteria’ LexA-binding sequences, and the other giving rise to the F. succinogenes and Myxococcus xanthus ones, in a transitional step towards the current ‘Gammaproteobacteria’ LexA box. The contrast between the results reported here and the phylogenetic data available in the literature suggests that, some time after its emergence as a distinct bacterial class, the ‘Alphaproteobacteria’ lost its vertically received lexA gene, but received later through lateral gene transfer a new lexA gene belonging to either a cyanobacterium or a bacterial species closely related to this phylum. This constitutes the first report based on experimental evidence of lateral gene transfer in the evolution of a gene governing such a complex regulatory network as the bacterial SOS system.
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The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression
Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAOΔmvaT, was constructed and characterized. The lysate of PAOΔmvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAOΔmvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.
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- Pathogens And Pathogenicity
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Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis
More LessEnterohaemorrhagic Escherichia coli (EHEC) cause acute gastroenteritis in humans that may be complicated by life-threatening systemic sequelae. The predominant EHEC serotype affecting humans in the UK and North America is O157 : H7 and infections are frequently associated with contact with ruminant faeces. Strategies to reduce the carriage of EHEC in ruminants are expected to lower the incidence of human EHEC infections; however, the molecular mechanisms underlying persistence of EHEC in ruminants are poorly understood. This paper reports the first comprehensive survey for EHEC factors mediating colonization of the bovine intestines by using signature-tagged transposon mutagenesis. Seventy-nine E. coli O157 : H7 mutants impaired in their ability to colonize calves were isolated and 59 different genes required for intestinal colonization were identified by cloning and sequencing of the transposon insertion sites. Thirteen transposon insertions were clustered in the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system required for the formation of attaching and effacing lesions on intestinal epithelia. A putative structural component of the apparatus (EscN) is essential for intestinal colonization; however, the type III secreted effector protein Map plays only a minor role. Other Type III secretion-associated genes were implicated in colonization of calves by E. coli O157 : H7, including z0990 (ecs0850), which encodes the non-LEE-encoded type III secreted effector NleD and the closely related z3023 (ecs2672) and z3026 (ecs2674) genes which encode homologues of Shigella IpaH proteins. We also identified a novel fimbrial locus required for intestinal colonization in calves by E. coli O157 : H7 (z2199-z2206; ecs2114-ecs2107/locus 8) and demonstrated that a mutant harbouring a deletion of the putative major fimbrial subunit gene is rapidly out-competed by the parent strain in co-infection studies. Our data provide valuable new information for the development of intervention strategies.
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Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS–ArlR two-component system of Staphylococcus aureus
More LessBacterial pathogens such as Staphylococcus aureus undergo major physiological changes when they infect their hosts, requiring the coordinated regulation of gene expression in response to the stresses encountered. Several environmental factors modify the expression of S. aureus virulence genes. This report shows that the expression of spa (virulence gene encoding the cell-wall-associated protein A) is down-regulated by high osmolarity (1 M NaCl, 1 M KCl or 1 M sucrose) in the wild-type strain and upregulated by novobiocin (a DNA gyrase inhibitor that relaxes DNA). A gyrB142 allele corresponding to a double mutation in the B subunit of DNA gyrase relaxed DNA and consequently induced spa expression, confirming that spa expression is regulated by DNA topology. Furthermore, in the presence of novobiocin plus 1 M NaCl, a good correlation was observed between DNA supercoiling and spa expression. The ArlS–ArlR two-component system is involved in the expression of virulence genes such as spa. Presence of an arlRS deletion decreased the effect of DNA supercoiling modulators on spa expression, suggesting that active Arl proteins are necessary for the full effect of DNA gyrase inhibitors and high osmolarity on spa expression. Indeed, evidence is provided for a relationship between the arlRS deletion and topological changes in plasmid DNA.
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Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions
More LessData are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the α-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), β-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.
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Characterization of the fibrinogen-binding surface protein Fbl of Staphylococcus lugdunensis
More LessThe fbl gene of Staphylococcus lugdunensis encodes a protein Fbl that is 58 % identical to the clumping factor A (ClfA) of Staphylococcus aureus. The fbl gene was present in eight clinical isolates of S. lugdunensis. When Fbl was expressed on the surface of Lactococcus lactis it promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution. Purified recombinant Fbl region A bound to immobilized fibrinogen in a dose-dependent manner and inhibited the adherence of both Fbl-expressing and ClfA-expressing strains of L. lactis to fibrinogen. Adherence of S. lugdunensis and L. lactis Fbl+ to immobilized fibrinogen was also inhibited by rabbit anti-Fbl region A antibodies and rabbit anti-ClfA region A antibodies, as well as by human immunoglobulin with a high level of anti-ClfA antibodies. Alignment of the A domains of CflA and Fbl revealed that all of the ClfA residues implicated in binding to the γ-chain of fibrinogen are conserved in Fbl. Nevertheless Fbl had a tenfold lower affinity for fibrinogen, suggesting that sequence differences that occur elsewhere in the protein, possibly in β-strand E of domain N2, affect ligand binding.
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σ B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment
More LessListeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor σ B, encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0·3 M NaCl) or acid (pH 4·5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes ΔsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that σ B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that σ B contributes to the ability of L. monocytogenes to cause foodborne infections.
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- Physiology
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Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58
More LessUsing the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.
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Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A–porin complexes
More LessRelease of colicin A was studied in Escherichia coli cells that differed in expressing the colicin A lysis protein (Cal). Pools of released and unreleased colicin A were harvested throughout colicin A induction. The amount of colicin A in each pool varied with the time of induction, allowing the definition of two sequential steps in colicin A release, one of which was dependent on Cal. Each step of colicin A release was differently affected in cells containing Cal mutants in which the N-terminal cysteine residue was substituted by either proline or threonine, preventing them from being acylated and matured. These Cal mutants were only observed in degP cells, indicating that the DegP protease cleaved the unacylated precursor of Cal. Cal was found in the insoluble fraction of the pools of released and unreleased colicin A together with the hetero-oligomers of colicin A and porins (colicins Au). The biogenesis of colicins Au was studied in temperature-sensitive secA and secY strains and found to be Sec-independent, indicating that they are formed by newly synthesized colicin A binding to mature porins already incorporated in the outer membrane. Cal is a lipoprotein similar to VirB7, a constituent of the type IV secretion system. It would interact with colicins Au to constitute the colicin A export machinery.
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2-Methylcitrate-dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein
More LessThe function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter.
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- Plant-Microbe Interactions
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Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization
The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6·5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes.
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